Vaccination with MHC-II restricted ApoB100 peptides to prevent atherosclerosis. Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries triggered by elevated levels of low density lipoprotein (LDL). Recent evidence shows that both innate and adaptive immune responses contribute to the disease. Using an in vitro screening assay, we found 34 peptides from mouse apolipoprotein (Apo)B100 that bind major histocompatibility complex (MHC)-II (I-Ab in Apoe-/- mice) with high affinity and tested 5 of them in vivo. We discovered that vaccination with I-Ab-binding ApoB100 peptides is strongly atheroprotective in the Apoe-/- mouse model of atherosclerosis. The reductions of atherosclerotic lesion size are associated with increased IL-10 expression in the aortas of vaccinated mice. Since we find elevated IL-10 in aortas of vaccinated mice, we hypothesize that immunization with ApoB100 peptides induces IL-10-producing Tregs and/or IL-10-producing macrophages. Specific aim 1 is to test which cell type is responsible for IL-10 production in aortas of vaccinated mice. We have crossed Apoe-/- mice to IL-10 reporter mice to identify the IL-10-producing cells. Cell type-specific IL-10 knockout mice (Il10fl/fl x LysM-Cre or x FoxP3- Cre) will identify the relevance of this proposed mechanism. Based on preliminary data, we hypothesize that Tregs afford protection from atherosclerosis. Specific aim 2 is to test the role o regulatory T cells (Tregs) in atheroprotection by ApoB100 peptides. We propose to define the antigen-specific CD4 T cells by surface phenotype and cytokine secretion, test their homing to atherosclerotic mouse aortas and study their interaction with dendritic cells (DCs) in atherosclerotic plaque by live cell multiphoton imaging. Based on preliminary data identifying 30 human ApoB100 peptides that bind many alleles of human MHC-II, we propose that ApoB100-based atheroprotective vaccination strategies are translatable to humans. Specific aim 3 is to translate atheroprotective vaccination to humans. We discovered one peptide that is human-to-mouse ApoB100 sequence-identical and binds to many human MHC-II alleles including DRB1*0101. We will test the effect of this sequence-identical ApoB100 peptide on atherosclerosis in existing mice transgenic for DRB1*0101, optimize vaccination regimens (adjuvant, frequency, dose) and evaluate the potential of tetramers and peptide- specific antibodies as biomarkers reporting successful vaccination. When the proposed work is completed, we will know (1) the role of IL-10 in vaccination-induced atheroprotection (2) the role of Tregs in atheroprotection and the mechanism of their homing and maintenance in the aorta and (3) whether this approach is indeed practical and translatable to humans. We already have an in vitro screening assay and propose to develop biomarkers to test for successful vaccination. Ultimately, a safe and effective atherosclerosis vaccine could have a very large national and world-wide public health impact.